The rapid expansion of new technologies for molecular diagnostics and tumor-targeted therapy has increased the need to develop highly specific targeting ligands for cell surface molecules that are expressed differentially in tumor cells or tissues.
Axl is a member of a receptor tyrosine kinases (RTK) family that also includes Dtk and Mer (Hafizi and Dahlbäck, 2006) and is activated by the growth factor, growth arrest specific 6 (GAS6). Ligand-induced stimulation of Axl mediates the activation of multiple downstream signaling pathways which play pivotal roles in regulating growth, proliferation and survival.
Axl was originally identified as a transforming gene in patients with chronic myelogenous leukemia (O'Bryan et al., 1991; Janssen et al., 1991). Subsequently Gash-Axl signaling has been implicated in a host of discrete cellular responses including cell survival, proliferation, migration, and adhesion (Linger et al., 2008). Overexpression of Axl has been associated with invasiveness and metastasis in a wide array of human cancers including lung (Shieh et al., 2005), prostate (Sainaghi et al., 2005), breast (Meric et al., 2002; Zhang et al., 2008), gastric (Wu et al., 2002) and pancreatic (Koorstra et al., 2009) cancers, renal cell carcinoma (Chung et al., 2003) as well as glioblastoma (Vajkoczy P, et al., 2006; Hutterer et al., 2008).
These data indicate that Axl signaling represents a novel target class for tumor therapeutic development.
An emerging wave of targeted therapeutic molecules against RTKs is composed of nucleic acid-based aptamers. They are short, structured single-stranded RNA or DNA ligands that bind with high affinity to their target molecules. Aptamers are isolated by the Systematic Evolution of Ligands by EXponential enrichment (SELEX) technology that since its first description in 1990 (Ellington and Szostak, 1990; Tuek and Gold, 1990), has yielded several high-affinity ligands of a wide variety of targets ranging from small chemical compounds to cells and tissues (Cerchia et al., 2002; Cerchia and de Franciscis, 2010). Aptamers are now emerging as promising molecules to target specific cancer epitopes in clinical diagnosis and therapy. Because of their high specificity and low toxicity, aptamers might be considered as the compounds of choice for in vivo cell recognition. In this perspective, nucleic acid aptamers represent a class of ligands that can rival antibodies for specificity and affinity for the target and is coupled to slow degradation kinetics and low toxicity. Furthermore, aptamers can be readily chemically modified by the addition of polyethylene glycol and other moieties to enhance their bioavailability and pharmacokinetics.
To date, only few inhibitors of Axl have been reported that are completely unrelated to the anti-Axl aptamer both from the structural and mode of action point of view:
1) small-molecule inhibitors, such as R428, that block the catalytic activities of Axl (Holland et al., 2010; Zhang et al., 2008);
2) an anti-Axl monoclonal antibody that blocks the ligand Gas6 binding to the receptor (Ye et al., 2010) proteins derived from the extracellular domain of Axl that inhibit its action by competing for ligand (GAS6) binding (International Patent application WO2008098139).
The present invention has identified an aptamer, GL21 52-85, that can solve the major problems related to the in vivo use of prior art inhibitors. GL21 52-85 aptamer is highly specific for the Axl receptor whereas R428 is effective not only on Axl but also on other tyrosine and serine/threonine kinases (i.e. Tie-2, Flt-1, Flt-3, Ret, Abl). Compared to anti-Axl antibodies, both antibodies and the anti-Axl aptamer have binding affinities in the low nanomolar range. However, the aptamer lacks immunogenicity, whereas antibodies in humans are significantly immunogenic, thus precluding repeat dosing unless they are “humanized” or produced fully human. RNA-based therapeutics are thus likely to be safer when repeated administrations are necessary. Further, the aptamer contains pyrimidines modified at the 2′-position, which render the RNA resistant to extracellular nucleases and even less immunogenic than natural RNA. Moreover, the aptamer can be readily chemically modified by the chemical addition of poly(ethylene glycol) (PEG) and other moieties to enhance bioavailability and pharmacokinetic properties. Because aptamers are synthesized by solid phase chemical synthesis, conjugation chemistry is possible at any position in the molecule at difference of proteins and peptides that can accept conjugation only on specific residues.
Further, GL21 52-85 offers several advantages over monoclonal antibodies due to its specificity and affinity for the target, slow degradation kinetics and low toxicity.